Fig 1. Environmental DNA marker development may be difficult or impossible when the target taxon’s taxonomy is unresolved or has poor concordance with true phylogeny.

Colored boxes show recognized taxa and black lines show the phylogenetic relationships among populations. (A) Taxon X includes multiple, divergent lineages. It is important that all of these lineages are sampled during marker validation to insure intraspecific generality. Even extensive sampling of a single lineage (red circle) is not sufficient and will lead to ascertainment bias and low marker generality. (B) Recognized taxa X and Y are not monophyletic (i.e., they are both paraphyletic groups), which may make distinguishing between them using sequence data impossible. (C) Recognized taxon X includes divergent lineages (polyphyly), but some of those lineages may also not be distinguishable from recognized taxon Y.